1. Field of the Invention
The present invention relates to baculovirus-expressed influenza antigens, in particular, to the influenza A and B viral nucleoprotein antigens expressed from Autographa californica nuclear polyhedrosis virus (AcNPV). The invention further relates to bioassays for the detection of influenza viral infections and to the use of such proteins in vaccines against influenza A or B virus.
2. Background Information
The nucleoprotein (NP) is the major protein component of the ribonucleoprotein core of influenza virions and is a type-specific antigen defining influenza viruses as either type A or type B. Recent studies have also identified the nucleoprotein antigen as a major target of the cross-reactive cytotoxic T-cell response against influenza viruses (Townsend et al., 1984, Journal of Experimental Medicine 160, 552-583); therefore, cellular immunity to NP may contribute to recovery from influenza infection (Wraith et al., 1987, Journal of General Virology 68, 433-440).
As antigenic variation of NPs occurs more slowly than does that of influenza surface glycoproteins (Van Wyke et al., 1980, Journal of Virology 35, 24-30), the NP is particularly useful for serodiagnostic tests for the detection of influenza infections in mammals. The test for measuring antibodies to NP has traditionally been a complement fixation (CF) test. However, the CF test has several drawbacks. For example, the CF test requires that a large number of biological reagents be standardized. In addition, the NP antigen preparations used in the CF test are likely to contain other virus-specific antigens. Serodiagnostic tests which overcome these drawbacks would be valuable tools for the detection of influenza infections.
Since the NP antigen is antigenically conserved, it is an ideal candidate for expression by recombinant DNA methodology as part of the development of a new generation of diagnostic assays for influenza virus infections. Viral antigens produced by recombinant DNA expression systems can provide an inexhaustible source of chemically defined material for use in serodiagnostic assays, experimental vaccines, and fundamental research. These techniques also eliminate the costs and potential hazards in the large-scale cultivation of pathogenic viruses. For example, the use of baculovirus-expressed Hantaan virus nucleoprotein as a diagnostic antigen has been reported recently (Schmalijohn et al., 1988, Journal of General Virology 69, 777-786). Further, the use of NP antigen produced in Escherichia coli (E. coli) in an experimental diagnostic assay for influenza A has been reported (Harmon et al., 1989, Journal of Medical Virology 24, 25-30). This assay was found to be superior to CF for influenza virus diagnosis.
The recently developed eucaryotic expression system using recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), should be useful for producing antigens for immunoassays for the serologic diagnosis of viral infections (Luckow et al., 1987, Bio/Technology 6, 47-55). Infecting insect cells (Spodoptera frugiperda) with such recombinant baculoviruses allows for the production of large amounts of antigen (Possee, R. D., 1986, Virus Research 5, 43-59). In addition, the baculovirus system has other important advantages over the commonly used methods of producing viral antigens. For example, with the baculovirus system the viral antigens are produced in cells that do not contain antigens that cross-react with antibodies in most human serum. Therefore, the purification of the antigen that is required for proteins expressed in bacterial and yeast systems may not be required with baculovirus-expressed antigens. Further, baculoviruses do not infect humans and can therefore be safely handled in large quantities.